Activating cAMP/PKA signaling in skeletal muscle suppresses the ubiquitin-proteasome-dependent proteolysis: implications for sympathetic regulation.

Although we have recently demonstrated that plasma catecholamines induce antiproteolytic effects on skeletal muscle (Graça FA, Gonçalves DAP, Silveira WA, Lira EC, Chaves VE, Zanon NM, Garófalo MAR, Kettelhut IC, Navegantes LCC. Am J Physiol Endocrinol Metab. 305: E1483-E1494, 2013), the role of the muscle sympathetic innervation and, more specifically, norepinephrine (NE) in regulating the ubiquitin (Ub)-proteasome system (UPS) remains unknown. Based on previous findings that chemical sympathectomy acutely reduces UPS activity, we hypothesized that muscle NE depletion induces adrenergic supersensitivity in rat skeletal muscles. We report that surgical sympathetic denervation (SDEN), a condition in which only muscle NE from both hindlimbs is depleted, transiently reduced the overall proteolysis and the UPS activity (∼25%) in both soleus and extensor digitorum longus muscles. This antiproteolytic response was accompanied by increased activity of adenylyl cyclase (112%), levels of cyclic adenosine monophosphate (cAMP; 191%), and the serine phosphorylation of cAMP response element-binding protein (32%). In extensor digitorum longus from normal rats, NE (10(-4) M) in vitro increased the levels of cAMP (115%) and the serine phosphorylation of both cAMP response element-binding protein (2.7-fold) and forkhead box class O1 transcription factor. Similar effects were observed in C2C12 cells incubated with forskolin (10 µM). In parallel, NE significantly reduced the basal UPS (21%) activity and the mRNA levels of atrophy-related Ub-ligases. Similar responses were observed in isolated muscles exposed to 6-BNZ-cAMP (500 µM), a specific PKA activator. The phosphorylation levels of Akt were not altered by SDEN, NE, forskolin or 6-BNZ-cAMP. Our results demonstrate that SDEN induces muscle adrenergic supersensitivity for cAMP leading to the suppression of UPS, and that the suppressive effects of NE on UPS activity and expression of Ub-ligases can be mediated by the activation of cAMP/PKA signaling, with the inhibition of forkhead box class O1 transcription factor.

Skeletal muscle expresses the extracellular cyclic AMP-adenosine pathway.

BACKGROUND AND PURPOSE: cAMP is a key intracellular signalling molecule that regulates multiple processes of the vertebrate skeletal muscle. We have shown that cAMP can be actively pumped out from the skeletal muscle cell. Since in other tissues, cAMP efflux had been associated with extracellular generation of adenosine, in the present study we have assessed the fate of interstitial cAMP and the existence of an extracellular cAMP-adenosine signalling pathway in skeletal muscle. EXPERIMENTAL APPROACH: cAMP efflux and/or its extracellular degradation were analysed by incubating rat cultured skeletal muscle with exogenous cAMP, forskolin or isoprenaline. cAMP and its metabolites were quantified by radioassay or HPLC, respectively. KEY RESULTS: Incubation of cells with exogenous cAMP was followed by interstitial accumulation of 5'-AMP and adenosine, a phenomenon inhibited by selective inhibitors of ecto-phosphodiesterase (DPSPX) and ecto-nucleotidase (AMPCP). Activation of adenylyl cyclase (AC) in cultured cells with forskolin or isoprenaline increased cAMP efflux and extracellular generation of 5'-AMP and adenosine. Extracellular cAMP-adenosine pathway was also observed after direct and receptor-dependent stimulation of AC in rat extensor muscle ex vivo. These events were attenuated by probenecid, an inhibitor of ATP binding cassette family transporters. CONCLUSIONS AND IMPLICATIONS: Our results show the existence of an extracellular biochemical cascade that converts cAMP into adenosine. The functional relevance of this extracellular signalling system may involve a feedback modulation of cellular response initiated by several G protein-coupled receptor ligands, amplifying cAMP influence to a paracrine mode, through its metabolite, adenosine.

Ectopic development of skeletal muscle induced by subcutaneous transplant of rat satellite cells.

The present study analyzes the ectopic development of the rat skeletal muscle originated from transplanted satellite cells. Satellite cells (10(6) cells) obtained from hindlimb muscles of newborn female 2BAW Wistar rats were injected subcutaneously into the dorsal area of adult male rats. After 3, 7, and 14 days, the transplanted tissues (N = 4-5) were processed for histochemical analysis of peripheral nerves, inactive X-chromosome and acetylcholinesterase. Nicotinic acetylcholine receptors (nAChRs) were also labeled with tetramethylrhodamine-labeled alpha-bungarotoxin. The development of ectopic muscles was successful in 86% of the implantation sites. By day 3, the transplanted cells were organized as multinucleated fibers containing multiple clusters of nAChRs (N = 2-4), resembling those from non-innervated cultured skeletal muscle fibers. After 7 days, the transplanted cells appeared as a highly vascularized tissue formed by bundles of fibers containing peripheral nuclei. The presence of X chromatin body indicated that subcutaneously developed fibers originated from female donor satellite cells. Differently from the extensor digitorum longus muscle of adult male rat (87.9 +/- 1.0 microm; N = 213), the diameter of ectopic fibers (59.1 microm; N = 213) did not obey a Gaussian distribution and had a higher coefficient of variation. After 7 and 14 days, the organization of the nAChR clusters was similar to that of clusters from adult innervated extensor digitorum longus muscle. These findings indicate the histocompatibility of rats from 2BAW colony and that satellite cells transplanted into the subcutaneous space of adult animals are able to develop and fuse to form differentiated skeletal muscle fibers.

Ectopic development of skeletal muscle induced by subcutaneous transplant of rat satellite cells

The present study analyzes the ectopic development of the rat skeletal muscle originated from transplanted satellite cells. Satellite cells (10(6) cells) obtained from hindlimb muscles of newborn female 2BAW Wistar rats were injected subcutaneously into the dorsal area of adult male rats. After 3, 7, and 14 days, the transplanted tissues (N = 4-5) were processed for histochemical analysis of peripheral nerves, inactive X-chromosome and acetylcholinesterase. Nicotinic acetylcholine receptors (nAChRs) were also labeled with tetramethylrhodamine-labeled alpha-bungarotoxin. The development of ectopic muscles was successful in 86 percent of the implantation sites. By day 3, the transplanted cells were organized as multinucleated fibers containing multiple clusters of nAChRs (N = 2-4), resembling those from non-innervated cultured skeletal muscle fibers. After 7 days, the transplanted cells appeared as a highly vascularized tissue formed by bundles of fibers containing peripheral nuclei. The presence of X chromatin body indicated that subcutaneously developed fibers originated from female donor satellite cells. Differently from the extensor digitorum longus muscle of adult male rat (87.9 ± 1.0 æm; N = 213), the diameter of ectopic fibers (59.1 æm; N = 213) did not obey a Gaussian distribution and had a higher coefficient of variation. After 7 and 14 days, the organization of the nAChR clusters was similar to that of clusters from adult innervated extensor digitorum longus muscle. These findings indicate the histocompatibility of rats from 2BAW colony and that satellite cells transplanted into the subcutaneous space of adult animals are able to develop and fuse to form differentiated skeletal muscle fibers.

Comparison of the efficacy of biodegradable and non-biodegradable scintillation liquids on the counting of tritium- and [14C]-labeled compounds.

The widespread use of H and 14C in research has generated a large volume of waste mixed with scintillation liquid, requiring an effective control and appropriate storage of liquid radioactive waste. In the present study, we compared the efficacy of three commercially available scintillation liquids, Optiphase HiSafe 3, Ultima-Gold AB (biodegradable) and Insta-Gel-XF (non-biodegradable), in terms of [14C]-glucose and [ H]-thymidine counting efficiency. We also analyzed the effect of the relative amount of water (1.6 to 50%), radioisotope concentration (0.1 to 100 nCi/ml), pH (2 to 10) and color of the solutions (samples containing 0.1 to 1.0 mg/ml of Trypan blue) on the counting efficiency in the presence of these scintillation liquids. There were few significant differences in the efficiency of 14C and H counting obtained with biodegradable or non-biodegradable scintillation liquids. However, there was an 83 and 94% reduction in the efficiency of 14C and H counting, respectively, in samples colored with 1 mg/ml Trypan blue, but not with 0.1 mg/ml, independent of the scintillation liquid used. Considering the low cost of biodegradable scintillation cocktails and their efficacy, these results show that traditional hazardous scintillation fluids may be replaced with the new safe biodegradable fluids without impairment of H and 14C counting efficiency. The use of biodegradable scintillation cocktails minimizes both human and environmental exposure to hazardous solvents. In addition, some biodegradable scintillation liquids can be 40% less expensive than the traditional hazardous cocktails.

Comparison of the efficacy of biodegradable and non-biodegradable scintillation liquids on the counting of tritium- and [14C]-labeled compounds

The widespread use of H and 14C in research has generated a large volume of waste mixed with scintillation liquid, requiring an effective control and appropriate storage of liquid radioactive waste. In the present study, we compared the efficacy of three commercially available scintillation liquids, Optiphase HiSafe 3, Ultima-GoldÕ AB (biodegradable) and Insta-Gel-XF (non-biodegradable), in terms of [14C]-glucose and [ H]-thymidine counting efficiency. We also analyzed the effect of the relative amount of water (1.6 to 50 percent), radioisotope concentration (0.1 to 100 nCi/ml), pH (2 to 10) and color of the solutions (samples containing 0.1 to 1.0 mg/ml of Trypan blue) on the counting efficiency in the presence of these scintillation liquids. There were few significant differences in the efficiency of 14C and H counting obtained with biodegradable or non-biodegradable scintillation liquids. However, there was an 83 and 94 percent reduction in the efficiency of 14C and H counting, respectively, in samples colored with 1 mg/ml Trypan blue, but not with 0.1 mg/ml, independent of the scintillation liquid used. Considering the low cost of biodegradable scintillation cocktails and their efficacy, these results show that traditional hazardous scintillation fluids may be replaced with the new safe biodegradable fluids without impairment of H and 14C counting efficiency. The use of biodegradable scintillation cocktails minimizes both human and environmental exposure to hazardous solvents. In addition, some biodegradable scintillation liquids can be 40 percent less expensive than the traditional hazardous cocktails.

Short- and long-term influences of calcitonin gene-related peptide on the synthesis of acetylcholinesterase in mammalian myotubes.

The present study analyses the short- (15 min - 2 h) and long-term (24 - 48 h) influences of calcitonin gene-related peptide (CGRP) on acetylcholinesterase (AChE) expression in the rat cultured skeletal muscle and the signal transduction events underlying CGRP actions. To assess the effect of CGRP on AChE synthesis, myotubes were pre-exposed to the irreversible AChE inhibitor diisopropyl fluorophosphate (DFP) and treated with CGRP or forskolin, an adenylyl cyclase (AC) activator. Treatment of myotubes with 1 - 100 nM CGRP for 2 h increased by up to 42% the synthesis of catalytically active AChE with a parallel increase in the intracellular cyclic AMP. The stimulation of AChE synthesis induced by CGRP was mimicked by direct activation of AC with 3 - 30 microM forskolin. In contrast, pre-treatment of cultures with 100 nM CGRP for 20 h reduced by 37% the subsequent synthesis of AChE, resulting in a 15% decrease in total AChE activity after 48 h CGRP treatment. Moreover, 24 h treatment of myotubes with 100 nM CGRP reduced by 54% the accumulation of cyclic AMP induced by a subsequent CGRP treatment. These findings indicate that, in skeletal muscle cells, CGRP modulates the AChE expression in a time-dependent manner, initially stimulating the enzyme synthesis through a cyclic AMP-dependent mechanism. The decreased AChE synthesis observed after long-term CGRP treatment suggests that CGRP signalling system is subject to desensitization or down-regulation, that might function as an important adaptative mechanism of the muscle fibre in response to long-term changes in neuromuscular transmission.

Evidence for activation of the tissue kallikrein-kinin system in nociceptive transmission and inflammatory responses of mice using a specific enzyme inhibitor.

The pharmacological activity of phenylacetyl-Phe-Ser-Arg-N-(2, 4-dinitrophenyl)-ethylenediamine (TKI), a tissue kallikrein specific inhibitor, was assessed using models of nociception and inflammation in mice. Injection of TKI (13.6 - 136 micromol kg(-1), i.p. or 41 - 410 micromol kg(-1), s.c.) produced a dose-related inhibition of the acetic acid-induced writhes (by 37 to 85% or 34 to 80%, respectively). The antinociceptive activity of TKI (41 micromol kg(-1), i.p.) was maximal after 30 min injection and lasted for 120 min. The effect was unaltered by pretreatment with naloxone (8.2 micromol kg(-1), s.c.) or bilateral adrenalectomy. TKI (41 and 136 micromol kg(-1), i.p.) produced a dose-related decrease of the late phase of formalin-induced nociception by 79 and 98%, respectively. At 136 micromol kg(-1), i.p., TKI also shortened the duration of paw licking in the early phase by 69%. TKI (41 and 136 micromol kg(-1), i.p.) also reduced the capsaicin-induced nociceptive response (by 51 to 79%). TKI (41 micromol kg(-1), i.p. or 410 micromol kg(-1), s.c.) reduced the oedematogenic response, from the second to the fifth hour after carrageenin injection by 36 to 30% or by 47 to 39%, respectively. Pretreatment with TKI (41 micromol kg(-1), i.p.) reduced the capsaicin-induced neurogenic inflammation in the mouse ear by 54%. It is concluded that TKI presents antinociceptive and antiinflammatory activities mediated by inhibition of kinin formation by tissue kallikrein in mice. The results also indicate that the tissue kallikrein-dependent pathway contributes to kinin generation in nociceptive and inflammatory processes in mice.

Testosterone control of endplate and non-endplate acetylcholinesterase in the rat levator ani muscle.

The time course of effects of castration (5-60 days) and testosterone treatment (15-60 days) of adult male rats were examined on the endplate (+EP) and non-endplate (-EP) acetylcholinesterase (AChE) of the androgen-dependent levator ani (LA) muscle. The thiocholine method was used to determine the enzyme activity. Castration caused LA muscle atrophy within 5 days but reduced the -EP and +EP AChE after 10 and 20 days, respectively. Following 30 days castration -EP and +EP AChE reached respectively 41% and 35% of control activity. Testosterone retrieval restored the control values of both muscle weight and total AChE after 15 and 60 days, respectively. Recovery of the +EP AChE preceded that of -EP AChE by 30 days. The results showed that in the rat LA muscle, +EP and -EP AChE depend on a continuous testosterone regulation that predominates at +EP region spreading thereafter to -EP region. Those data suggest a hormone regulation of AChE exerted indirectly through the synthesis and release of neurotrophic substances.

Globular and asymmetric forms of acetylcholinesterase in the rat levator ani muscle.

The endplate (+EP) and non-endplate (-EP) distribution of molecular forms of acetylcholinesterase (AChE) was compared in the dimorphic levator ani and diaphragm muscles from adult male rats. Enzyme activity was measured by the thiocholine method and AChE forms were separated on the basis of solubility in sodium phosphate buffer of different ionic strength. For the dimorphic levator ani muscle, total AChE activity was 324.6 +/- 18.9 nmol ASCh hydrolyzed min-1 muscle-1, 90% of which was globular and predominated in the -EP region (78%). The asymmetric forms were almost exclusively detected in the +EP region (9%). In diaphragm muscle, total AChE activity was 176.7 +/- 11.0 units; 66% was mainly globular and located in the -EP region (56%); the asymmetric forms (34%) were either in -EP (11%) or +EP (23%) regions. Thus, a greater proportion of globular form was present in the dimorphic levator ani muscle than in diaphragm muscle. In view of the control exerted by testosterone on dimorphic muscles, it is suggested that the greater synthesis of the globular form in the levator ani occurs under the trophic influence of testosterone.

Pharmacological screening of Ageratum conyzoides L. (Mentrasto).

The pharmacological activities of a water extract (WE) of Ageratum conyzoides L, a plant popularly known for its analgesic and anti-inflammatory properties, were studied in vivo and in vitro preparations. Oral administration (p.o.) of the water extract (WE, 0.1 to 5 g/kg) to rats and mice induced quietness and reduced the spontaneous motility. The sleeping time induced by sodium pentobarbital (50 mg/kg, i.p.) in mice was not altered by previous treatment with WE (2 g/kg, p.o.). The same treatment did not influence the paw edema induced by carrageenan or dextran, nor did it reduce the chronic paw edema induced by complete Freund's adjuvant or formaldehyde in rats. The tail flick response in immersion test and writhings induced by 0.8% acetic acid in mice were not altered by WE either. In isolated guinea-pig ilea WE (0.4 to 4 mg/ml) did not alter the EC50 values of histamine or acetylcholine, but reduced the maximal response to the agonists by 20 to 50%. WE (0.01 to 10 mg/ml) produced tonic contractions of the ileal smooth muscle proportional to the doses, reaching a maximum of 75% relatively to the maximum obtained with histamine. Those contractions were blocked by diphenhydramine (10 nM) and reduced by 32% in presence of atropine (10 nM). The results indicated that oral treatment of rodents with A. conyzoides L neither reduced the inflammatory edema nor did it decrease the reaction to pain stimuli. In vitro the extract presented an unexpected histamine-like activity characteristic of a partial agonist. The results did not confirm the popular medicinal indications of the plant.

Globular and asymmetric forms of acetylcholinesterase in the rat levator ani muscle

The endplate (+EP) and non-endplate (-EP) distribution of molecular formas of acetylcholinesterase (AChE) was compared in the dimorphic levator ani and disphragm muscles from adult male rats. Enzyme activity was measured by the thicholine method and AchE forms were separated on the basis of solubility in sodium phosphate buffer of different ionic strenght. For the dimorphic levator ani muscle, total AchE activity was 342.6 ñ 18.9 nmol ASCh hydrolyzed min-1 muscle-1, 90% of which was globular and predominated in the-EP region (78%). The asymmetric forms were almost exclusively detected in the +EP region (9%). In diaphragm muscler, total AChE activity was 176.7 ñ 11.0 units; 66% was mainly globular and located in the-EP region (56%); the asymmetric forms (34%) were either in -EP (11%) or + EP (23%) regions. Thus, a greater proportion of globular form was presented in the dimorphic levator ani muscle than in diaphragm muscle. In view of the control exerted by testosterone on dimorphic muscles, its is suggested that the grater synthesis of the globular form in the levator ani occurs under the trophic influence of testosterone

Early and late influences of testosterone on acetylcholinesterase activity of skeletal muscles from developing rats.

1. The influence of perinatal and pubertal gonadal androgens on acetylcholinesterase (AChE) activity was studied in the hormone-sensitive levator ani (LA) and extensor digitorum longus (EDL) muscles of adult male rats (105 days). 2. The hormone was withdrawn by gonadectomy at various ages and the effects on AChE and weight were compared with those induced by chronic denervation of both muscles from adult rats. 3. Gonadectomy of infantile (2-30 days) rats prevented LA muscle growth, and reduced total AChE activity to values similar to those found in denervated muscles (15% of control). The EDL muscles were slightly affected and only in rats castrated on the 2nd postnatal day. 4. When the rats were castrated at puberty (45 days), LA muscle weight and total AChE activity were reduced to 20% and 18% of control values, respectively. 5. Gonadectomy of adult (60 and 75 days) rats led to atrophy of the LA muscle (to 29% of control) and reduced the total AChE activity (to 40% of control). 6. AChE activity per unit weight was reduced by 30% in rats castrated from 5 to 20 days of age, and increased by 30% in both LA and EDL muscles from rats castrated in adulthood. Gonadectomy before puberty prevented total AChE in the LA from increasing above the levels detected in chronically denervated muscles. 7. Gonadectomy after puberty reduced total AChE of the LA but never to the extent caused by muscle denervation. 8. It is concluded that testosterone regulates AChE in the LA by early priming of the motoneuron and by pubertal stimulation of enzyme synthesis, the synthesis being dependent on intact innervation.

Trophic control of cholinesterase activity in a testosterone-dependent muscle of the rat. II. Effects of testosterone administration.

The effects of testosterone on the weight, protein content, and acetylcholinesterase (AChE) activity were investigated in the hormone-dependent levator ani and nondependent extensor digitorum longus and soleus muscles from normal or castrated male rats. In either group some muscles were also chronically denervated. Testosterone propionate treatment (3 mg/week for 2 weeks, s.c.) of normal rats increased the weight and protein content of the levator ani, respectively, by 19% and 63%; the muscle AChE was not affected. Protein content, but not the weight of the normal extensor digitorum longus and soleus was also increased after testosterone; AChE was reduced by 20% in the extensor digitorum longus and unaltered in the soleus. In castrated rats, testosterone reversed the levator ani atrophy and slowed down the decay of AChE, but it did not restore the normal enzyme activity. Testosterone did not prevent the atrophy and AChE decrease induced by denervation of either muscle. The weight and protein content of the denervated levator ani from castrated rats were increased by testosterone to the values found in denervated muscles from normal rats; AChE in the same muscles was not increased. The results confirm that separate mechanisms regulate protein synthesis and AChE in the rat levator ani. AChE is mainly regulated by neural factors which in turn appear to be influenced by circulating androgens. Similar hormonal influence on the muscle AChE was not detected in the extensor digitorum longus and soleus muscles.

Trophic control of cholinesterase activity in a testosterone-dependent muscle of the rat: effects of castration and denervation.

The effects of testosterone withdrawal and chronic denervation on muscle weight and acetylcholinesterase (AChE) activity were studied in the hormone-sensitive levator ani muscle of the rat. Castration of adult male rats for 7 to 60 days caused a linear decrease of the weight, protein content, and AChE activity of the muscle, which stabilized after 30 days. Muscle weight and protein content decreased 2.3% per day. The total AChE activity decreased 7 days later 3.2% per day, reaching 37% of control at day 30. AChE activity per unit weight was increased in all castrated groups. Muscle weights and AChE activity of the extensor digitorum longus and soleus muscles were not altered after castration. Denervation of all three muscles caused 50% reduction of the muscle weight and protein content after 15 days. Total AChE activity decayed exponentially with a rate of 0.12 per day to 15 to 18% of control values. AChE activity per unit weight in the denervated muscles was always lower than in the control muscles. Combined castration and denervation intensified only the levator ani protein loss. The different onset and time course of the effects induced by castration and denervation indicate distinct mechanisms involved in the trophic control of muscle proteins and AChE activity. Chronic muscle denervation decreased total AChE activity to 15% of normal, whereas castration reduced the enzyme to 40% of the control values. The results indicate that neuronal and hormonal influences on AChE activity of the levator ani are not additive but overlap.